Antibiotic resistance has severely limited treatment options for many potentially life-threatening bacterial infections and has become an enormous problem for human health. This situation has only been exacerbated by the dearth of new antibiotics in development. Therefore, this proposal has the long-term goal of developing new drugs as potential therapeutics to treat bacterial infections. Our strategy relies on inhibiting bacterial virulence factors rather than indiscriminately killing all bacteria. We have chosen specialized secretion systems as our target as they are utilized by a wide range of pathogens to cause disease. One major class of secretion systems are called type IV (T4SS) and include both plasmid transfer system, which are often involved in transfer of antibiotic resistance, and adapted conjugation systems used by a number of intracellular pathogens. One such pathogen, Legionella pneumophila, uses a T4SS named Dot/Icm to survive and replicate within alveolar macrophages, thereby causing a bacterial form of pneumonia called Legionnaires' Disease. The L. pneumophila Dot/Icm T4SS is a remarkable apparatus as it injects almost three hundred toxins into host cells. As a result, it is required for virulence and represents an ideal target fo antimicrobials. We propose to identify and characterize inhibitors of T4SSs using cyclic peptides generated by SCICLOPPS (split-intein circular ligation of peptides and proteins) technology. This strategy has the major advantage of being able to generate massive libraries consisting of millions of different compounds. Based on our extensive knowledge about the L. pneumophila Dot/Icm system, we have developed several methods that will enable the rapid selection of cyclic peptide inhibitors. The combination of high complexity libraries and the use of selections (rather than screens) to identify T4SS inhibitors has vast potential to generate lead compounds in the quest for novel targeted antibacterial therapies. We anticipate that these compounds will likely function as inhibitors not just of L. pneumophila but also of other pathogens that contain related type IV secretion systems such as Coxiella burnetii.